BioScience Trends. 2009; 3(2): Department of Anesthesiology, Changzheng Hospital, The Second Military Medical University, Shanghai, China; 3

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1 48 Original Article Protective effect of anti-intercellular adhesion molecule-1 antibody on global cerebral ischemia/reperfusion injury in the rat Jianping Cao 1, Xueyin Shi 2, *, Weiyan Li 3, Jian Liu 3, Xiaoyong Miao 1, Jia Xu 1 1 Department of Anesthesiology, Hospital No. 455 of the PLA, Shanghai, China; 2 Department of Anesthesiology, Changzheng Hospital, The Second Military Medical University, Shanghai, China; 3 Department of Anesthesiology, Jinling Hospital, School of Medicine, Nanjing University, Nanjing, China. Summary The present study aimed to clarify the protective effect of administration of an antiintercellular adhesion molecule-1 (ICAM-1) antibody (1A29) on neurological damage after global cerebral ischemia/reperfusion in rats. Global cerebral ischemia/reperfusion was produced by four-vessel occlusion for 30 min followed by reperfusion for 24 h. Animals were randomly divided into four groups: PC group (n = 10), PI group (n = 10), PR group (n = 10), and PM group (n = 10). Rats in the PC group were administered isotype-matched control antibody at a dose of 1 mg/kg IV. Rats in the PI group, PR group, and PM group were infused with 1A29 at a dose of 1 mg/kg IV before ischemia, upon reperfusion, and 4 h into reperfusion, respectively. All animals were sacrificed after reperfusion for 24 h. Cerebral sections were stained with hematoxylin and eosin for histological evaluation. The brain wet-to-dry ratio and neurological deficits were evaluated. In comparison to the PC group, the counts of polymorphonuclear leukocytes (PMNLs) and macrophages (MΦ) decreased significantly in the PI, PR, and PM groups (P < 0.01). In comparison to the control antibody group, the brain wet-to-dry ratio and the percent infarct volume were significantly reduced in rats receiving 1A29 antibody (P < 0.05 and P < 0.01, respectively). In comparison to the PC group, with a median neurological score of 2.5, mild deficits were noted in the PI, PR, and PM groups (median neurological scores were 1.6 to 1.8) (P < 0.05). 1A29 antibody decreased the counts of PMNLs and MΦ and the neurological score and it reduced the brain wet-to-dry ratio and the infarct volume, suggesting that anti-icam-1 antibody provides neuroprotection after global cerebral ischemia/reperfusion injury in rats. Keywords: Global cerebral, ischemia/reperfusion, intercellular adhesion molecule-1, antibody, rat 1. Introduction Inflammatory response and oxidative stress are known to exacerbate the damage caused by acute cerebral ischemia/reperfusion injury (1,2). Cytokines formed immediately after ischemia stimulate the expression of adhesion molecules on endothelial cells and leukocytes, leading to leukocyte adherence and extravasation into brain parenchyma (3,4). Extravasated polymorphonuclear leukocytes (PMNLs) release *Address correspondence to: Dr. Xueyin Shi, Department of Anesthesiology, Changzheng Hospital, The Second Military Medical University, 415 Fengyang Road, Shanghai 20003, China. shixueyin1128@yahoo.com.cn reactive oxygen species and lipid peroxidation products and promote blood-brain barrier disruption, vascular plugging, edema, and cerebral infarction (5,6). The migration of leukocytes into injured tissue is regulated in part by a specific cell-surface integrin known as the CD18 receptor complex (7). Intercellular adhesion molecule-1 (ICAM-1) is a cell surface glycoprotein that is expressed on vascular endothelium and other cells. ICAM-1 expression facilitates leukocyte adhesion to endothelium (8). Previous studies demonstrated that drugs designed to inhibit recruited leukocytes/microglia markedly curtailed inflammation and oxidative stress-related apoptosis and consequently provided neuroprotection in cerebral I/R injury (9,10). Previous studies showed a significant decrease in middle cerebral artery

2 49 occlusion (MCAO)-induced brain damage in ICAM-1 knockout mice and anti-icam-1 antibody-treated rats (11). Treatment with the anti-icam-1 antibody reduces neurological deficits after spinal cord injury and embolic stroke in the rabbit (12). In rodents, hypothermic neuroprotection against focal ischemia is associated with attenuation of ICAM-1 induction and PMNL infiltration (13,14). Thus, these data support the hypothesis that neutrophils contribute to ischemic cell damage and blocking of ICAM-1 expression reduces ischemic cell damage. However, the optimal point in time for antibody administration has yet to be determined. The present study evaluated the efficacy of anti-icam-1 antibody used at various times to prevent infarct development and neurological deficiency after global cerebral ischemia/reperfusion injury in adult rats. 2. Material and Methods 2.1. Animals Sprague-Dawley rats (weight, 180 to 200 g; n = 40) used in this study were cared for in accordance with the NIH guidelines for the ethical use of laboratory animals. The Research Animal Resources and Care Committee of Nanjing University approved the surgical procedures. Animals fasted for 8 h before surgery and were allowed free access to water. All animals were anesthetized with ketamine (80 mg/kg, i.p.) Four-vessel occlusion model A four-vessel occlusion model as described earlier was used to induce global cerebral ischemia/reperfusion (14). Under ketamine anesthesia, a pin 0.5 mm in diameter was inserted through each alar foramen and both vertebral arteries were cauterized and permanently occluded. Through a ventral mid-cervical incision, each carotid artery was isolated and a 9-0-nylon ligature was looped around it. After 24 h, global brain ischemia/reperfusion was induced by traction on both carotid ligatures for 30 min and then loosening of both ligatures. During ischemia/reperfusion, body temperature (37 C to 38 C) and cranial temperature (36 C to 37 C) were maintained at the physiological level Grouping Animals were randomly divided into four groups: PC group (n = 10), PI group (n = 10), PR group (n = 10), and PM group (n = 10). Rats in all groups were subjected to ischemia for 30 min and reperfusion for 24 h. Rats in the PC group were administered isotypematched control antibody at a dose of 1 mg/kg IV. Rats in the PI group were infused with 1A29 at a dose of 1 mg/kg IV before ischemia. Rats in the PR group and PM group were infused with 1A29 at the same dosage upon reperfusion and 4 h into reperfusion, respectively. Antibody to rat ICAM-1, designated 1A29 (15), reacts with the 85- to 89-kD epitope present on cytokineactivated rat endothelial cells. The endotoxin level of the anti-icam-1 antibody is less than 0.35 eu/mg. The control antibody has an endotoxin level of less than 1.0 eu/mg Determination of degrees of brain injury All rats were sacrificed after reperfusion for 24 h. Tissues of the right cerebrum were processed and embedded in paraffin, and 4 μm-thick paraffin sections were stained with hematoxylin-and-eosin for histopathological evaluation. Six random high-double views (magnification, 100) were taken to count PMNLs and macrophages (MΦ). The volume of the ischemic lesion was computed by the numeric integration of data from 12 to 14 sections with respect to the sectional interval, as described earlier (16). The infarct volume was corrected to account for edema and shrinkage due to processing. The injury volumes were corrected using the following formula: corrected injury volume = contralateral hemisphere volume (ipsilateral hemisphere volume measured injury volume). The indirect method for calculating infarct volume, in which the intact area of the ipsilateral hemisphere was subtracted from the area of the contralateral hemisphere, was used. The infarct volume is presented as the percentage of the infarct lesion of the contralateral hemisphere Measurement of the brain wet-to-dry ratio After the rats were sacrificed, cerebral tissues of the left hemisphere were removed and immediately weighed. The cerebral tissues were dried in an oven at 80 C for 12 h and reweighed. The brain wet-to-dry ratios were obtained by dividing the mass of the initial specimen by the mass of the dried specimen Neurological evaluation Global cerebral ischemia/reperfusion induced neurological deficit was evaluated on a 6-point scale following 1 day of reperfusion (before the animals were sacrificed) by an investigator blinded to the study groups, as described earlier (17). A score of 0 suggests no neurological deficit (normal), 1 suggests a mild neurological deficit (e.g. failure to fully extend the right forepaw), 2 suggests a moderate neurological deficit (e.g. circling to the right), 3 suggests a severe neurological deficit (e.g. falling to the right), and 4 suggests a very severe neurological deficit (e.g. failing to walk spontaneously and having a reduced level of consciousness).

3 Statistical analysis All values are presented as mean ± standard error. Statistical evaluation was performed with the use of ANOVA followed by an unpaired t test. Significance was indicated by P < 0.05, and a high level of significance was indicated by P < Results 3.1. Infiltration of PMNLs and MΦ Table 1 shows the PMNLs and MΦ counts in the 1A29 groups and the groups treated with control antibody. In comparison to the PC group, the counts of PMNLs and MΦ decreased significantly in the PI, PR, and PM groups (P < 0.01). No significant difference in the counts of PMNLs and MΦ was detected among the 1A29 groups Wet-to-dry ratio of injured cerebral tissue Disruption of the blood-brain barrier was assessed by measuring water and fluid content in the brain. In Table 1. PMNLs and MΦ infiltration on cerebral in various groups Groups PC PI PR PM PMNLs 12.3 ± ± 2.0 * 6.0 ± 1.8 * 7.2 ± 2.4 * Infiltration a,b MΦ 2.1 ± ± 0.3 * 1.1 ± 0.6 * 1.4 ± 0.2 * a Data are represented as the mean ± SE; b Comparison with PC group, * P < comparison to the control antibody group, the brain wetto-dry ratio was significantly reduced in rats receiving 1A29 antibody (P < 0.05) (Figure 1) Determination of infarct volume Table 2 shows representative infarct areas in rats with control antibody and 1A29 infusion. Total infarct volume did not differ significantly among the groups. In comparison to the control antibody group, the percent infarct volume decreased significantly in the 1A29-treated group after global cerebral ischemia/reperfusion injury (P < 0.01) Effect of anti-icam-1 antibody on neurological deficits The neurological deficits analyzed at 24-h reperfusion were severe in the PC group, with a median neurological score of 2.5, compared with mild deficits in the PI, PR, and PM groups (median neurological scores were 1.6 to 1.8) (Figure 2). Table 2. Absolute hemisphere and lesion volumes and percent lesion volume of the contralateral hemisphere in various groups Groups PC PI PR PM Hemisphere ± ± ± ± 18.8 Volumes (mm 3 ) a,b Lesion ± ± 11.7 * ± 12.4 * ± 9.6 * % Lesion volume b 60.6 ± ± 4.8 * 33.5 ± 7.5 * 38.8 ± 3.9 * a Data are represented as the mean ± SE; b Comparison with PC group, * P < * 4 3 BWDR (%) PC PI PR PM Figure 1. Brain water content in rats with global cerebral ischemia/ reperfusion injury. Brain water content was determined by measuring the brain wet-to-dry ratio (BWDR) in rats with global cerebral ischemia/reperfusion injury. In comparison to the PC group, BWDR was significantly reduced in the PI, PR, and PM groups. * P < Data were analyzed with the unpaired t test and are shown as mean ± SE. Neuroscore PC PI PR PM Individual rats Figure 2. Individual neurological scores upon 24 h of reperfusion after global cerebral ischemia in rats with control antibody and rats with 1A29 infusion (n = 10 per group). In comparison to the PC group, with a median neurological score of 2.5, mild deficits were noted in the PI, PR, and PM groups (median neurological scores were 1.6 to 1.8). * P < 0.05.

4 51 4. Discussion The current data indicate that intravenous administration of an anti-icam-1 antibody (1A29) significantly reduces the volume of infarcts and the brain wet-todry ratio and it decreases the subject's neurological score and the counts of PMNLs and MΦ. However, no significant difference in the above indices was observed among the 1A29 groups. The protective effect of anti-icam-1 antibody against ischemic/reperfusion injury is attributed to the blockage of leukocyte adhesion, transendothelial migration, and improvement of blood flow during reperfusion (18). Ferulic acid provides neuroprotection against oxidative stress-related apoptosis after cerebral ischemia/reperfusion injury by inhibiting ICAM-1 mrna expression in rats (19). Previous findings by the current authors revealed a new mechanism of hypothermia brain protection via inhibition of ICAM-1 expression and blocking of PMNL and MΦ infiltration in a rat global cerebral I/R injury model (14). Studies have shown that prevention of ICAM-1 protein expression by antisense infusion significantly decreases transient focal ischemia induced infarct size and neurological deficits (20,21). However, the choice of using antisense versus antibodies depends on the state of the patient. Antisense can be used as a preventive measure to bind to mrna and inhibit ICAM-1 protein formation. Anti-ICAM-1 antibody may be more appropriate during ischemia/reperfusion injury by binding to ICAM-1 protein and halting its action. The present study supports the contention that anti- ICAM-1 antibody administered either before ischemia or after reperfusion has a neuroprotective effect. The anti-icam-1 antibody is effective in reducing ischemic cell damage when administered during the reperfusion period and specifically 4 h after the initiation of reperfusion. This has positive implications for use of this form of therapeutic intervention in a clinical environment, where delayed intervention may be needed. Further study is required to determine the last point after reperfusion at which this therapy can be used. In conclusion, the administration of the anti- ICAM-1 antibody significantly reduced global cerebral ischemic/reperfusion injury, regardless of whether it was before ischemia or after reperfusion. References 1. Shin DH, Bae YC, Kim-Han JS, Lee JH, Choi IY, Son KH, Kang SS, Kim WK, Han BH. Polyphenol amentoflavone affords neuroprotection against neonatal hypoxicischemic brain damage via multiple mechanisms. J Neurochem. 2006; 96: Iadecola C, Alexander M. Cerebral ischemia and inflammation. Curr Opin Neurol. 2001; 14: Khan M, Elango C, Ansari MA, Singh I, Singh AK. Caffeic acid phenethyl ester reduces neurovascular inflammation and protects rat brain following transient focal cerebral ischemia. J Neurochem. 2007; 102: Imai H, Graham DI, Masayasu H, Macrae IM. Antioxidant ebselen reduces oxidative damage in focal cerebral ischemia. Free Radic Biol Med. 2003; 34: Zoppo GJ, Becker KJ, Hallenbeck JM. Inflammation after stroke: is it harmful? Arch Neurol. 2001; 58: Ding Y, Young CN, Li J, Luan X, Clark JD, Diaz FG. Reduced inflammatory mediator expression by prereperfusion infusion into ischemic territory in rats: a real-time polymerase chain reaction analysis. Neurosci Lett. 2003; 353: Caimi G, Canino B, Ferrara F, Montana M, Musso M, Porretto F, Carollo C, Catania A, Lo Presti R. Granulocyte integrins before and after activation in acute ischaemic stroke. J Neurol Sci. 2001; 186: Arumugam TV, Salter JW, Chidlow JH, Ballantyne CM, Kevil CG, Granger DN. Contributions of LFA-1 and Mac-1 to brain injury and microvascular dysfunction induced by transient middle cerebral artery occlusion. Am J Physiol Heart Circ Physiol. 2004; 287: H2555-H Kao TK, Ou YC, Kuo JS, Chen WY, Liao SL, Wu CW, Chen CJ, Ling NN, Zhang YH, Peng WH. Neuroprotection by tetramethylpyrazine against ischemic brain injury in rats. Neurochem Int. 2006; 48: Storini C, Rossi E, Marrella V, Distaso M, Veerhuis R, Vergani C, Bergamaschini L, De Simoni MG. C1-inhibitor protects against brain ischemiareperfusion injury via inhibition of cell recruitment and inflammation. Neurobiol Dis. 2005; 19: Frijns CJ, Kappelle LJ. Inflammatory cell adhesion molecules in ischemic cerebrovascular disease. Stroke. 2002; 33: Zhang RL, Chopp M, Jiang N, Tang WX, Prostak J, Manning AM, Anderson DC. Anti-intercellular adhesion molecule-1 antibody reduces ischemic cell damage after transient but not permanent middle cerebral artery occlusion in the Wistar rat. Sroke. 1995; 26: Wang GJ, Deng HY, Maier CM, Sun GH, Yenari MA. Mild hypothermia reduces ICAM-1 expression, neutrophil infiltration and microglia/monocyte accumulation following experimental stroke. Neuroscience. 2002; 114: Gao JP,Xu JG, Li WY, Liu J. Influence of selective brain cooling on the expression of ICAM-1 mrna and infiltration of PMNLs and monocytes/macrophages in rats suffering from global brain ischemia/reperfusion injury. BioScience Trends. 2008; 2: Tamatani T, Miyasaka M. Identification of monoclonal antibody reactive with the rat homologue of ICAM-1, and evidence for differential involvement of ICAM-1 in the adherence of resting versus activated lymphocytes to high endothelial cells. Int Immunol. 1993; 2: Raghavendra Rao VL, Dogan A, Bowen KK, Dempsey RJ. Ornithine decarboxylase knockdown exacerbates transient focal cerebral ischemia-induced neuronal damage in rat brain. J Cereb Blood Flow Metab. 2001; 21: Rao VL, Dogan A, Todd KG, Bowen KK, Kim BT, Rothstein JD, Dempsey RJ. Antisense knockdown of the glial glutamate transporter GLT-1, but not the neuronal

5 52 glutamate transporter EAAC1, exacerbates transient focal cerebral ischemia-induced neuronal damage in rat brain. J Neurosci. 2001; 21: Zhang RL, Chopp M, Li Y, Zaloga C, Jiang N, Jones ML, Miyasaka M, Ward PA. Anti-ICAM-1 antibody reduces ischemic cell damage after transient middle cerebral artery occlusion in the rat. Neurology. 1994; 44: Chin YC, Shan YS, Nou YT, Tin YH, Su YC, Ching LH. Ferulic acid provides neuroprotection against oxidative stress-related apoptosis after cerebral ischemia/ reperfusion injury by inhibiting ICAM-1 mrna expression in rats. Brain Res. 2008; 1209: Rao VL, Dogan A, Bowen KK, Todd KG, Dempsey RJ. Antisense knockdown of the glial glutamate transporter GLT-1 exacerbates hippocampal neuronal damage following traumatic injury to rat brain. Eur J Neurosci. 2001; 13: Vemuganti R, Dempsey RJ, Bowen KK. Inhibition of intercellular adhesion molecule-1 protein expression by antisense oligonucleotides is neuroprotective after transient middle cerebral artery occlusion in rat. Stroke. 2004; 35: (Received February 13, 2009; Accepted February 23, 2009)

Imaging ischemic strokes: Correlating radiological findings with the pathophysiological evolution of an infarct

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